The search for improving and extending the capabilities of optical analysis have long involved considerations of the substrate on which the specimen is supported during the analysis.
In the case of biological material, use has been made of polymeric substrates, in particular, porous substrates also referred to as “membranes” and “matrices,” to immobilize the material while the material undergoes genetic analysis or is used for cell or protein research. Historically, porous matrices were first created as filters, to separate particulates contained within a liquid. In the process, a number of porous polymeric matrices were identified to have strong binding affinity for a number of bio-polymers. These matrices became the substrates of choice for cytochemistry and bio-polymer studies, especially where radioactive labels were employed.
The ability of nitrocellulose membranes (also referred to as “cellulose nitrate”) to serve as substrates to bind single stranded DNA, i.e., to immobilize DNA, was demonstrated by Nirenberg in 1965 in flow-through assays. Such membranes were commonly formed using fibrous cellulose as a starting material.
Cellulose, to which nitrocellulose is related, is formed as a chain of glucose units, which is the universal building material for living cells. Nitrocellulose membranes benefit in this regard by relationship to cellulose, and have been commonly used substrates because of their molecular binding properties. The membranes have been used to bind cells, bio-polymers, proteins, genetic material and nucleic acids, as well as serving as substrates for non-biological chemicals.
The use of micro-porous polymeric membranes, in particular, nitrocellulose, for blotting bio-molecules from electrophoretically separated molecules was developed by Southern for DNA-DNA interactions. The technique is commonly called “Southern” blotting in honor of the developer. The other compass directions have been developed. “Western” blotting is a technique that has been employed to immobilize protein on an immobilizing substrate for protein-protein interactions.
Southern's need was for a method to identify the separated zones in electrophoretic separation. The blotting method employed micro-porous nitrocellulose to specifically identify the electrophoretically separated zones.
A brief outline of the original Southern blotting technique may help understand the general function of the nitrocellulose substrate:                A sample, in this case containing DNA, is separated on a gel media by electrophoresis and is denatured by treatment with sodium hydroxide.        A micro-porous nitrocellulose membrane is placed over the gel.        Blotter paper is placed over the membrane to absorb the water from the gel and a weight is added on top. The weight forces the water and separated molecules into the micro-porous membrane as the gel collapses beneath the membrane. This leaves an image on the membrane comprised of the separated bio-molecules.        DNA from the zones is bound non-specifically to the micro-porous nitrocellulose membrane.        The nitrocellulose membrane is washed and blocked by diffusional methods.        For performing an assay, the nitrocellulose membrane is then incubated with a solution of a known labeled DNA.        If the DNA added is an exact match to a zone of the DNA immobilized from the electrophoretic separation, the labeled DNA will bind and the zone will be labeled.        After successive washings, a visual image of the labeled zones then is prepared using X ray film if a radioactive label were used, thus identifying the zones.        
Following the original development of Southern's techniques, in an effort to increase throughput, a trend developed to replace radioactive tracers with fluorescent tags, with the stimulated fluorescent emissions being imaged by optics. It was noticed, however, that available porous polymeric membranes, themselves, exhibited fluorescent emission over a wide spectral range. This fluorescent emission, as background noise, limited the use of polymeric membranes in fluorescent studies of proteins. While nitrocellulose membranes have been identified as one of the least offenders, still, when used as a substrate material, nitrocellulose has been found to have objectionable fluorescence that has limited both the accuracy of detection and the throughput of assays.
In the case of DNA, despite a continuing desire to employ polymeric membranes such as nitrocellulose, a way around the fluorescence problem was found, by spotting arrays on glass or quartz slides, that have relatively little background emission, using a layer of non-polymeric silane or GAP, and other such materials as adhesion promoters to which the biopolymer is directly bound. These adhesion promoter materials, despite their own significant problems, such as difficultly in obtaining a uniform thickness, noise contribution, and reactivity, have permitted significant success with small molecules. No similar technique has existed that is as effective for protein molecules, which are approximately 1000 times larger than DNA. Resort, still, has often been made to membranes of considerable thickness of porous nitrocellulose or other self-fluorescing polymeric immobilizing material, the material either being self-supporting or backed by a support. In the case of micro-porous nitrocellulose on a backing such as a microscope slide, typically the nitrocellulose has been at least 10 micron in thickness, and its self-fluorescence has remained a limiting factor for assay throughput. The significance to biology and to the clinician of the need to conduct higher throughput, large scale assays of protein arrays is discussed for instance in Chin et al., U.S. Pat. No. 6,197,599.
New insights are presented here about the substrates on which many of the known protein assays can be conducted. These insights lead broadly to techniques that increase throughput and achieve higher accuracy imaging of fluorescently- or luminenscently-labeled proteins and other bio-materials for large scale assays for research and for clinical diagnosis.